G. Ascone

FRI0049 Fc gamma receptor iv enhances bone erosion in experimental arthritis by promoting influx of pmns

Background FcγRs are involved in regulation of synovial activation and bone destruction during immune complex (IC)-mediated arthritis. The balance between activating FcγRs (FcγRI,III and IV) and inhibiting FcγRII determines synovial activation. Here we investigated the particular role of activating FcγRIV in bone erosion in IC-mediated antigen induced arthritis (AIA) by comparing FcγRI,II,III,IV-/- mice, FcγRI,II,III-/- mice and wild type controls (WT). Objectives To investigate the role of FcγRIV in bone erosion during experimental arthritis. Methods AIA was induced by injection of mBSA into knee joints of mice previously immunized with mBSA/CFA. Joint inflammation, bone destruction, number of TRAP+ osteoclasts and S100A8/A9 positive cells was determined using histology and immunohistochemistry. In vitro osteoclastogenesis was assessed using TRAP staining. Results Seven days after induction of AIA, we observed decreased inflammation and bone erosion in the knee joints of FcγRI,II,III,IV-/- mice compared to WT. The ability of bone marrow cells of FcγRI,II,III,IV-/- mice to differentiate into osteoclasts in vitro was comparable to the one of WT controls. Moreover, we observed comparable numbers of TRAP+ osteoclasts on the bone surface of FcγRI/II/III/IV-/- and WT arthritic mice, suggesting that the observed decrease in bone erosion is mainly caused by a reduced osteoclast activity, rather than decreased osteoclast number. However, in contrast to FcγRI/II/III/IV-/-, AIA induction in knee joints of FcγRI/II/III -/- resulted in increased bone erosion and inflammation compared to WT, highlighting the possible crucial role of FcγRIV in the pathology. Interestingly, the number of PMNs infiltrated in the knee joint of FcγRI/II/III-/- resulted increased, whereas it was decreased in the knee joints of FcγRI/II/III/IV-/- compared to their WT controls. This observation suggests that particularly FcγRIV is involved in regulating influx of PMNs. PMNs are potent producers of alarmins S100A8/A9 which are described to promote osteoclast activity. In line the number of S100A8/A9 positive cells in synovium was increased in FcγRI/II/III-/- while decreased in FcγRI/II/III/IV-/-, compared to their WT control. Conclusions FcγRIV promotes bone erosion in AIA by enhancing influx of PMNs within the synovium. PMNs are potent producers of S100A8/A9 which has been described to induce osteoclast activity. Disclosure of Interest None declared

S100A8/A9, a potent serum and molecular imaging biomarker for synovial inflammation and joint destruction in seronegative experimental arthritis

BackgroundSeronegative joint diseases are characterized by a lack of well-defined biomarkers since autoantibodies are not elevated. Calprotectin (S100A8/A9) is a damage-associated molecular pattern (DAMP) which is released by activated phagocytes, and high levels are found in seronegative arthritides. In this study, we investigated the biomarker potential of systemic and local levels of these S100 proteins to assess joint inflammation and joint destruction in an experimental model for seronegative arthritis.MethodsSerum levels of S100A8/A9 and various cytokines were monitored during disease development in interleukin-1 receptor antagonist (IL-1Ra)–/– mice using ELISA and multiplex bead-based immunoassay, and were correlated to macroscopic and microscopic parameters for joint inflammation, bone erosion, and cartilage damage. Local expression of S100A8 and S100A9 and matrix metalloproteinase (MMP)-mediated cartilage damage in the ankle joints were investigated by immunohistochemistry. In addition, local S100A8 and activated MMPs were monitored in vivo by optical imaging using anti-S100A8-Cy7 and AF489-Cy5.5, a specific tracer for activated MMPs.ResultsSerum levels of S100A8/A9 were significantly increased in IL-1Ra–/– mice and correlated with macroscopic joint swelling and histological inflammation, while serum levels of pro-inflammatory cytokines did not correlate with joint swelling. In addition, early serum S100A8/A9 levels were prognostic for disease outcome at a later stage. The increased serum S100A8/A9 levels were reflected by an increased expression of S100A8 and S100A9 within the ankle joint, as visualized by molecular imaging. Next to inflammatory processes, serum S100A8/A9 also correlated with histological parameters for bone erosion and cartilage damage. In addition, arthritic IL-1Ra–/– mice with increased synovial S100A8 and S100A9 expression showed increased cartilage damage that coincided with MMP-mediated neoepitope expression and in vivo imaging of activated MMPs.ConclusionsExpression of S100A8 and S100A9 in IL-1Ra–/– mice strongly correlates with synovial inflammation, bone erosion, and cartilage damage, underlining the potential of S100A8/A9 as a systemic and local biomarker in seronegative arthritis not only for assessing inflammation but also for assessing severity of inflammatory joint destruction.

S100A8/A9 increases the mobilization of pro-inflammatory Ly6Chigh monocytes to the synovium during experimental osteoarthritis

BackgroundMonocytes are dominant cells present within the inflamed synovium during osteoarthritis (OA). In mice, two functionally distinct monocyte subsets are described: pro-inflammatory Ly6Chigh and patrolling Ly6Clow monocytes. Alarmins S100A8/A9 locally released by the synovium during inflammatory OA for prolonged periods may be dominant proteins involved in stimulating recruitment of Ly6Chigh monocytes from the circulation to the joint. Our objective was to investigate the role of S100A8/A9 in the mobilization of Ly6Chigh and Ly6Clow monocytic populations to the inflamed joint in collagenase-induced OA (CiOA).MethodS100A8 was injected intra-articularly to investigate monocyte influx. CiOA was induced by injection of collagenase into knee joints of wild-type C57BL/6 (WT), and S100a9-/- mice. Mice were sacrificed together with age-matched saline-injected control mice (n = 6/group), and expression of monocyte markers, pro-inflammatory cytokines, and chemokines was determined in the synovium using ELISA and RT-qPCR. Cells were isolated from the bone marrow (BM), spleen, blood, and synovium and monocytes were identified using FACS.ResultsS100A8/A9 was highly expressed during CiOA. Intra-articular injection of S100A8 leads to elevated expression of monocyte markers and the monocyte-attracting chemokines CCL2 and CX3CL1 in the synovium. At day 7 (d7) after CiOA induction in WT mice, numbers of Ly6Chigh, but not Ly6Clow monocytes, were strongly increased (7.6-fold) in the synovium compared to saline-injected controls. This coincided with strong upregulation of CCL2, which preferentially attracts Ly6Chigh monocytes. In contrast, S100a9-/- mice showed a significant increase in Ly6Clow monocytes (twofold) within the synovium at CiOA d7, whereas the number of Ly6Chigh monocytes remained unaffected. In agreement with this finding, the Ly6Clow mobilization marker CX3CL1 was significantly higher within the synovium of S100a9-/- mice. Next, we studied the effect of S100A8/A9 on release of Ly6Chigh monocytes from the BM into the circulation. A 14% decrease in myeloid cells was found in WT BM at CiOA d7. No decrease in myeloid cells in S100a9-/- BM was found, suggesting that S100A8/A9 promotes the release of myeloid populations from the BM.ConclusionInduction of OA locally leads to strongly elevated S100A8/A9 expression and an elevated influx of Ly6Chigh monocytes from the BM to the synovium.

Genetic modification of ER‐Hoxb8 osteoclast precursors using CRISPR/Cas9 as a novel way to allow studies on osteoclast biology

Osteoclasts are cells specialized in bone resorption. Currently, studies on murine osteoclasts are primarily performed on bone marrow–derived cells with the use of many animals and limited cells available. ER‐Hoxb8 cells are conditionally immortalized monocyte/macrophage murine progenitor cells, recently described to be able to differentiate toward functional osteoclasts. Here, we produced an ER‐Hoxb8 clonal cell line from C57BL/6 bone marrow cells that strongly resembles phenotype and function of the conventional bone marrow–derived osteoclasts. We then used CRISPR/Cas9 technology to specifically inactivate genes by biallelic mutation. The CRISPR/Cas9 system is an adaptive immune system in Bacteria and Archaea and uses small RNAs and Cas nucleases to degrade foreign nucleic acids. Through specific‐guide RNAs, the nuclease Cas9 can be redirected toward any genomic location to genetically modify eukaryotic cells. We genetically modified ER‐Hoxb8 cells with success, generating NFATc1−/− and DC‐STAMP−/− ER‐Hoxb8 cells that lack the ability to differentiate into osteoclasts or to fuse into multinucleated osteoclasts, respectively. In conclusion, this method represents a markedly easy highly specific and efficient system for generating potentially unlimited numbers of genetically modified osteoclast precursors.

Fcγ receptor-mediated influx of S100A8/A9-producing neutrophils as inducer of bone erosion during antigen-induced arthritis

BackgroundOsteoclast-mediated bone erosion is a central feature of rheumatoid arthritis (RA). Immune complexes, present in a large percentage of patients, bind to Fcγ receptors (FcγRs), thereby modulating the activity of immune cells. In this study, we investigated the contribution of FcγRs, and FcγRIV in particular, during antigen-induced arthritis (AIA).MethodsAIA was induced in knee joints of wild-type (WT), FcγRI,II,III−/−, and FcγRI,II,III,IV−/− mice. Bone destruction, numbers of tartrate-resistant acid phosphatase-positive (TRAP+) osteoclasts, and inflammation were evaluated using histology; expression of the macrophage marker F4/80, neutrophil marker NIMPR14, and alarmin S100A8 was evaluated using immunohistochemistry. The percentage of osteoclast precursors in the bone marrow was determined using flow cytometry. In vitro osteoclastogenesis was evaluated with TRAP staining, and gene expression was assessed using real-time PCR.ResultsFcγRI,II,III,IV−/− mice showed decreased bone erosion compared with WT mice during AIA, whereas both the humoral and cellular immune responses against methylated bovine serum albumin were not impaired in FcγRI,II,III,IV−/− mice. The percentage of osteoclast precursors in the bone marrow of arthritic mice and their ability to differentiate into osteoclasts in vitro were comparable between FcγRI,II,III,IV−/− and WT mice. In line with these observations, numbers of TRAP+ osteoclasts on the bone surface during AIA were comparable between the two groups. Inflammation, a process that strongly activates osteoclast activity, was reduced in FcγRI,II,III,IV−/− mice, and of note, mainly decreased numbers of neutrophils were present in the joint. In contrast to FcγRI,II,III,IV−/− mice, AIA induction in knee joints of FcγRI,II,III−/− mice resulted in increased bone erosion, inflammation, and numbers of neutrophils, suggesting a crucial role for FcγRIV in the joint pathology by the recruitment of neutrophils. Finally, significant correlations were found between bone erosion and the number of neutrophils present in the joint as well as between bone erosion and the number of S100A8-positive cells, with S100A8 being an alarmin strongly produced by neutrophils that stimulates osteoclast resorbing activity.ConclusionsFcγRs play a crucial role in the development of bone erosion during AIA by inducing inflammation. In particular, FcγRIV mediates bone erosion in AIA by inducing the influx of S100A8/A9-producing neutrophils into the arthritic joint.

03.11 Apolipoprotein e stimulates inflammation and joint destruction during antigen-induced arthritis (aia)

Background Rheumatoid arthritis (RA) is a chronic inflammatory disease largely driven by immune complexes and their interaction with FcγRs on synovial macrophages. In RA, joint destruction has been associated with high levels of cholesterol (LDL) which become oxidised (oxLDL) within the inflamed synovium. Apolipoprotein E (ApoE) regulates LDL levels and its absence strongly elevates LDL in the serum. In this study we investigated the role of ApoE in inflammation and joint destruction during antigen-induced arthritis (AIA). Materials and methods Experimental arthritis was induced by injection of mBSA into the right knee joint of ApoE-/- and wild type (WT) mice previously immunised with mBSA/CFA. Joint swelling was measured by 99m Technecium (99mTc) uptake and expressed as a ratio of the uptake in the right (injected) and left (non injected) knee joint. Serum mBSA antibody titer was measured by ELISA. WT BM-MΦ were stimulated for 24 hours in vitro with or without 50 µg/ml oxLDL and the FcγRs mRNA expression was measured by qPCR. Joint inflammation and damage were measured by histology using an arbitrary scale from 0 to 3. Results ApoE-/- mice showed significantly less joint swelling at day 1, 3 and 7 after AIA induction compared to WT controls (21%, 17%, 18% lower, respectively). Serum mBSA antibody levels (total IgG, IgG1, IgG2a and IgG2b) were comparable between WT and ApoE-/-mice. LDL serum levels were significantly higher in ApoE-/- mice and LDL/oxLDL was found within synovial macrophages. At day 21, histology showed less inflammatory cells within the synovium and joint cavity (22% and 44% lower, respectively) in the ApoE-/- mice compared to WT controls. WT BM-MΦ stimulated with oxLDL displayed a significant down-regulation of mRNA levels of FcγR I and FcγR II when compared to their non stimulated controls (1,7 and 2,3 fold change, respectively). Joint destruction was significantly reduced in the ApoE-/- mice, as indicated by the reduction of chondrocyte death (32% reduction) and bone erosion (25% reduction from 1.5±0.2 to 1.1±0.1). Conclusions ApoE stimulates joint destruction during AIA by lowering LDL/oxLDL levels, thereby promoting Fcγ- mediated macrophage activation within the synovium.

A1.12 Local experimental osteoarthritis induces systemic changes in monocyte populations regulated by S100A8/A9

Inflammation is increasingly recognised to be involved in osteoarthritis (OA) pathology. In response to pro-inflammatory cytokines, monocytes can be recruited from the bone marrow (BM) where monocyte chemoattractant protein-1 (MCP-1) is a key molecule that drives monocyte efflux via binding with the C-C chemokine receptor type 2 (CCR2). In mice, two functionally distinct monocyte populations are described: pro-inflammatory Ly6C-high monocytes (CCR2high) and patrolling Ly6C-low monocytes (CCR2low). The objectives of our study are to investigate the systemic effects of locally induced OA on BM monocyte populations and their recruitment to the OA joint in collagenase induced OA (CiOA), and the role of the alarmins S100A8/A9 in that. CiOA was induced in C57BL/6 mice by unilateral-articular collagenase-injection and were sacrificed at day 7, 21 and 42, together with age-matched naive mice (n = 6/group), and synovial mRNA expression of several pro-inflammatory cytokines were measured. During CiOA, the absolute amount of cells in the BM per femur was measured and the mRNA expression of BM MCP-1 and CCR2 was determined. Cells from BM, blood and synovial tissue were isolated and analysed by FACS. Monocyte subsets were identified as (B220/CD90/CD49b/NK1.1/Ly6G)lowCD11bhigh(F4/80/MHCII/CD11c)low and further distinguished by their Ly6C expression. In addition, we investigated synovitis in S100A9-/-mice during CiOA. Synovial expression of IL-1β, IL-6, TNF-α, S100A8 and S100A9 was increased at day 7, but only expression of S100A8 and S100A9 remained high until day 21. Local induction of CiOA resulted in systemic effects within the BM showing decreased cell numbers at day 7 and 21 (15% and 14% respectively). Concurrently, the expression of MCP1 at day7 in BM was increased 3.3-fold, suggesting increased BM-efflux. Relative number of BM-Ly6C-high monocytes was increased at day 7 (164%), being reflected by increased CCR2 expression (2.8-fold). This suggests a specific regulation of BM Ly6C-high monocytosis and recruitment during CiOA. During the course of CiOA increased number of Ly6C-high monocytes were observed in the synovium: 398% at day 7 and 510% at day 42, compared to naïve mice. These effects may be caused by the sustained S100A8/A9 levels, we therefore investigated synovitis in S100A9-/- mice during CiOA. The number of cell layers and cell influx in the synovium of S100A9-/- mice was decreased compared to C57BL/6 mice Local induction of OA induces systemic release of BM-derived cells and increased Ly6C-high monocyte populations systemically and locally in the synovium, a process that may be regulated by the sustained release of S100A8/A9 from the synovium. Disclosure of Interest None declared.

OP0214 S100A8/A9 Produced Locally during Experimental Osteoarthritis Induces Local and Systemic Changes in Monocyte Populations

Background The etiology of osteoarthritis (OA) is multi-factorial, and is mainly driven by an activated synovium wherein inflammation plays an important role. In response to pro-inflammatory cytokines, monocytes can be recruited from the bone marrow (BM) to the site of inflammation. In mice, two functionally distinct monocyte populations are described: pro-inflammatory Ly6C-high monocytes (C-C chemokine receptor type 2 (CCR2)high) and patrolling Ly6C-low monocytes (CCR2low). In the BM, monocyte chemoattractant protein-1 (MCP-1) is a key molecule that drives monocyte efflux via binding with CCR2. Objectives The objectives of our study are to investigate the systemic effects of locally induced OA on BM monocyte populations and their recruitment to the OA joint in collagenase induced OA (CiOA), and the role of the alarmins S100A8/A9 in that. Methods CiOA was induced by unilateral-articular collagenase-injection in C57BL/6 mice. At day 7, 21 and 42, mice were sacrificed together with age-matched naive mice (n=6/group), and synovial mRNA expression of several pro-inflammatory cytokines were measured. During CiOA and control conditions, the absolute amount of cells in the BM per femur was measured and the mRNA expression of BM MCP-1 and CCR2 was determined. Cells from BM, blood and synovial tissue were isolated and analyzed by FACS. Monocyte subsets were identified as (B220/CD90/CD49b/NK1.1/Ly6G)lowCD11bhigh(F4/80/MHCII/CD11c)low and further distinguished by their Ly6C expression. In addition, we investigated synovitis in S100A9–/– mice during CiOA. Results Expression of the pro-inflammatory cytokines IL-1β, IL-6, TNF-α, S100A8 and S100A9 in the synovium were increased at day 7, but only expression of S100A8 and S100A9 remained high until day 21. Local induction of CiOA resulted in systemic effects within the BM as shown by the decrease in cell numbers at day 7 and 21 (15% and 14% respectively). Concurrently, the expression of MCP1 at day 7 in BM was increased (3.3-fold), suggesting an increased efflux of BM-cells. Relative number of BM-Ly6C-high monocytes was increased at day 7 (164%), being reflected by increased CCR2 expression (2.8-fold). This suggests a specific regulation of BM Ly6C-high monocytosis and recruitment during CiOA and emphasizes the systemic effects following CiOA. During the course of CiOA increased numbers of Ly6C-high monocytes were also observed in the synovium: 398% at day 7 and 510% at day 42, compared to naïve mice. These effects may be caused by the sustained S100A8/A9 levels; we therefore investigated synovitis in S100A9–/– mice during CiOA. The number of cell layers and cell influx in the synovium of S100A9–/– mice was decreased compared to C57BL/6 mice. Conclusions Local induction of OA induces systemic release of BM-derived cells and increased Ly6C-high monocyte populations systemically and locally in the synovium, a process that may be regulated by the sustained release of S100A8/A9 from the synovium. Disclosure of Interest None declared

OP0210 Apolipoprotein E Aggravates Inflammation and Joint Destruction in Murine Antigen-Induced Arthritis

Background Rheumatoid arthritis (RA) is a chronic inflammatory disease characterized by severe bone destruction which has been associated with altered lipid metabolism. Apolipoprotein E (Apo E) is a lipoprotein crucial in lipid metabolism, mainly produced by macrophages. Moreover ApoE has been recently described as an important anti-inflammatory mediator regulating innate immunity and bone turnover. Objectives In the present study we investigated the role of Apo E in inflammation and bone destruction during antigen-induced arthritis (AIA). Methods Experimental arthritis (AIA) was induced by injection of 60 μg mBSA into the right knee joint of ApoE–/– and wild type (WT) control mice previously immunized with mBSA/CFA. Joint swelling was measured by uptake of 99mTechnecium (99mTc) and expressed as a ratio of the uptake in right (injected) and the left (non injected) knee joint. Humoral immunity (mBSA antibody titer) was measured by ELISA. Joint inflammation and bone erosion were measured by histological analysis using an arbitrary scale from 0 to 3. TRAP+ cells were determined using immunohistochemistry. Results ApoE–/– mice showed significantly less joint swelling at day 1, 3 and 7 after AIA induction compared to WT controls (21%, 17%, 18% lower, respectively). Serum mBSA antibody levels (total IgG, IgG1, IgG2a and IgG2b) are comparable between the two immunized mouse strains. At day 21, histology of the knee joints showed less infiltration of inflammatory cells (25% lower) and decreased bone erosion in the ApoE–/– mice compared to WT controls (25% lower from 1.5±0.2 to 1.1 ±0.1). In line with that, ApoE–/– mice showed a reduction of the number of osteoclasts present at the area of resorption within the arthritic knee joints (36% lower from 20±4 osteoclasts/section in WT mice to 12±5 in ApoE–/– mice), as measured by image analysis of TRAP staining. Conclusions ApoE aggravates bone destruction within the knee joints during AIA by increasing influx of inflammatory cells within the synovium and elevating the number of resorbing osteoclasts on the bone surface. Disclosure of Interest None declared

A1.30 Locally induced experimental osteoarthritis results in a system bone marrow shift towards a pro-inflammatory monocyte subpopulation

Background and objectives A significant role for inflammation during osteoarthritis (OA) is increasingly recognised, which involves the recruitment of immune cells, including monocytes, towards the inflamed synovium. In mice two functionally distinct monocyte populations are described; Ly6C-high monocytes, which express CCR2 and are considered pro-inflammatory and Ly6C-low monocytes, which express CX3CR1 and are suggested to be involved in repair processes. These monocytes arise from the bone marrow (BM) where monocyte chemoattractant protein-1 (MCP-1) is a key molecule that drives the monocyte efflux. As second tissue from which monocytes may originate is the spleen. The aim of this study is to investigate systemic effects of locally induced OA on BM and splenic monocyte subpopulations and the recruitment of these monocytes to the OA joint synovium in collagenase induced osteoarthritis (CiOA). Materials and methods CiOA was locally induced in C57Bl/6 mice by injection of collagenase in the right knee joint. Seven and 42 days after induction, mice (n = 6) were sacrificed, together with age-matched naive C57Bl/6 mice. Cells from BM, spleen, blood and knee synovial tissue were isolated and analysed by FACS. Ly6C-high monocytes were identified as (B220/CD90/CD49b/NK1.1/Ly6G)lowCD11bhigh(F4/80/MHCII/CD11c)lowLy6Chigh and Ly6C-low monocytes as (B220/CD90/CD49b/NK1.1/Ly6G)lowCD11bhigh(F4/80/MHCII/CD11c)lowLy6Clow. BM expression of MCP-1, CCR2 and CX3CR1 mRNA was determined at day 7 and 42 by q-PCR. Results In naive synovium few monocytes were present (1012 ± 925 Ly6C-high and 621 ± 488 Ly6C-low monocytes), which is likely an artefact of residual blood. At day 7 after CiOA induction, the number of Ly6C-high and -low monocytes in the OA synovium was 420% and 300% increased, respectively, compared to naive synovium. In blood, monocyte subpopulations were not changed, but in BM, the number of Ly6C-high monocytes was 160% increased, while Ly6C-low monocytes were decreased by 170%. Furthermore, expression of MCP-1 and CCR2 was increased by 3.2 and 2.8 times, while CX3CR1 expression remained unchanged. Even at day 42 increased levels of both monocyte subpopulations were observed in the OA synovium (Ly6C-high; 280% and Ly6C-low; 220%). In the BM, no change in both monocyte subpopulations was observed anymore as well as no change in expression of MCP-1, CCR2 and CX3CR. In spleen no changes in Ly6C-high or -low monocytes were observed throughout the course of CiOA, indicating no role for splenic monocytes in the recruitment of monocytes towards the OA synovium. Conclusions These data indicate that compared to naïve synovium, increased numbers of both Ly6C-high and -low monocytes are present in the OA synovium throughout the course of CiOA, but that a systemic effect on the BM monocyte subpopulations and their efflux is only observed in the early phase of OA. In the BM a clear skew towards a pro-inflammatory monocyte subset is visible, indicating that locally induced OA may also be systemically regulated.